Light-dependent activation of rod transducin by pineal opsin

Abstract

The pineal gland expresses a unique member of the opsin family (P- opsin; Max, M., McKinnon, P. J., Seidenman, K. J., Barrett, R. K., Applebury, M. L., Takahashi, J. S., and Margolskee, R. F. (1995) Science 267, 1502- 1506) that may play h role in circadian entrainment and photo-regulation of melatonin synthesis. To study the function of this protein, an epitope- tagged P-opsin was stably expressed in an embryonic chicken pineal cell line. When incubated with 11-cis-retinal, a light-sensitive pigment was formed with a A(max) at 462 ± 2 nm. P-opsin bleached slowly in the dark (t 1/4 = 2 h) in the presence of 50 mM hydroxylamine. Purified P-opsin in dodecyl maltoside activated rod transducin in a light-dependent manner, catalyzing the exchange of more than 300 mol of GTPγS (guanosine 5'-O-(3-thiotriphosphate))/mol of P-opsin. The initial rate for activation (75 mol of GTPγS bound/mol of P- opsin/min at 7 μM) increased with increasing concentrations of transducin. The addition of egg phosphatidylcholine to P-opsin had little effect on the activation kinetics; however, the intrinsic rate of decay in the absence of transducin was accelerated. These results demonstrate that P-opsin is an efficient catalyst for activation of rod transducin and suggest that the pineal gland may contain a rodlike phototransduction cascade.